A variety of chemical methods for converting N-protected amino acids, including the C-terminal amino acid residues of peptides, to a corresponding thiohydantoin (TH) have been proposed. TH formation is useful, for example, in C-terminal amino acid sequencing, where a C-terminal amino acid of a peptide is sequentially (a) converted to its corresponding TH, (b) cleaved from the remaining peptide, and (c) identified in term of the attached amino acid, e.g., by HPLC. Another use of such reaction methods is in preparing amino acid TH compounds for use as standards, e.g., in C-terminal sequencing.
The conversion of N-protected amino acids to corresponding amino acid TH's was first proposed in 1911 by Johnson et al. (1911) and in 1926 by Schlack and Kumpf (Schlack et al., 1926). A number of improvements to this early method have since been proposed (see, e.g., Stark, 1968; Boyd et al., U.S. Pat. No. 5,051,368; Hawke, 1987; Hawke, U.S. Pat. No. 4,837,165; Hawke et al., U.S. Pat. No. 5,049,507; Inglis; Miller, 1988; and Miller, U.S. Pat. No. 4,935,494).
Stark's method for making 2-thiohydantoins, employing acetic anhydride as activating agent, often results in low yields and in complicating side reactions. Similarly, the method of Hawke et al. (U.S. Pat. No. 5,049,507), which employs an acyl-isothiocyanate moiety for generating the thiohydantoin, suffers from many side reactions.
Recent efforts to improve thiohydantoin-based methods have focused on improving the efficiency of forming the C-terminal activated ester. For example, Boyd et al. (U.S. Pat. No. 5,051,368) have proposed a method of forming a C-terminal thiohydantoin that employs N-substituted ketenimines, generated by Woodward's Reagent K, to form the activated ester. However Reagent K is not soluble in organic solvents, and the reaction of the ketenimine intermediate (which is unstable in solution) with the polypeptidyl C-terminal carboxylate must usually be carried out slowly at room temperature.
Kenner et at. (1953), and more recently, Bailey et al. (U.S. Pat. No. 5,180,807) have used phosphoroisothiocyanatidate for preparing C-terminal thiohydantoins. Although the reagent used in the method performs the dual roles of carboxyl activation and thiodantoin formation, the reagent is inherently unstable and results in low TH yields.
A further problem in forming C-terminal thiohydantoins concerns the reactivity of the non-terminal carboxy groups appearing on the side-chains of aspartic and glutamic acid. Typically, these internal carboxy groups are derivatized along with the C-terminal carboxy. If these modified residues later appear as the C-terminal residue in the sequencing process, they frequently interfere with sequencing by giving rise to alternative side products during thiodantoin formation, or they are difficult or impossible to distinguish from other residues after cleavage.